Tendons are frequently injured, and unfortunately have poor innate healing capacity that significantly disrupts human motion. Limited treatment options motivate the need for novel regenerative therapies and tissue engineered tendon replacements. Our long-term goal is to generate functional tendon replacements that match the structure and mechanical function of native tendons by enhancing tendon formation in scaffolds seeded with stem cells. A common tissue engineering approach is to seed mesenchymal stem cells (MSCs) in extracellular matrix-derived scaffolds, such as collagen sponges. A majority of these approaches have applied mechanical stimuli and growth factors to the MSC-seeded scaffolds to direct differentiation of MSCs toward the tendon lineage (tenogenesis) and to enhance tissue formation. Unfortunately, these strategies have not resulted in an engineered tendon replacement that recapitulates the aligned collagen fiber structure and mechanical properties of tendon. Understanding the mechanisms that control scaffold remodeling by MSCs may be the missing detail needed to direct functional tendon formation in vitro. An additional challenge is controlling tenogenic differentiation of the MSCs. Matrix metalloproteinases (MMPs) are potent regulators of extracellular matrix remodeling, but it is unknown how MMPs impact tenogenic differentiation and tendon formation by MSCs. Also unknown are the mechanisms of MMP regulation by cyclic tensile loading, a common strategy used to guide tenogenic differentiation by MSCs. In other cell types, hypoxia inducible factor (HIF)-1? has been found to be activated by mechanical loading and is implicated as a regulator of MMP production. However, the HIF-1? pathway has not been investigated as a mechanotransducer or regulator of MMP production in MSCs. To address these missing details in the regulation of MMPs and tenogenic differentiation, our study will determine the relationship between MMPs and tenogenesis by MSCs. Our innovative approach will explore the potential for MMPs to impact tenogenesis and identify a potential mechanism of their mechanoregulation. This project will test the hypothesis that increased MMP activity enhances tenogenesis in vitro, and production of MMPs by MSCs is mechanoregulated by the HIF-1? pathway. In the first aim, we will determine how exogenous MMP treatment influences tendon formation and tenogenesis of MSCs in collagen sponges, and how inhibiting MMP activity alters the MSC response to mechanical stimuli. Results of these experiments will determine the role of MMPs in regulating tenogenic differentiation markers and tendon formation by MSCs. In the second aim, we will explore the impact of mechanotransduction by the HIF-1? pathway on MMP production by MSCs. These experiments will identify the HIF-1? pathway as a mechanoregulator of MMP production by MSCs. Results of this project provide insight into the mechanisms responsible for MMP activity-dependent regulation of tissue formation and stem cell tenogenesis, which have wide-reaching implications for tendon tissue engineering and regeneration.